Current techniques for preserving the heart for transplantation remain inadequate and damage to the vascular endothelium contributes significantly to the morbidity and mortality of heart recipients. Endothelial cells are at risk during heart preservation because they are directly exposed to rapid cooling and near-00C temperatures during vascular flushing and cold storage and to rapid rewarming and re-oxygenation during reperfusion, conditions that generate oxidative injury. The long-term goal is to understand the molecular basis of hypothermia-mediated cardiac tissue damage, and use this knowledge to develop improved therapies for protecting the heart during cardiothoracic surgery and transplantation. Preliminary studies showed that human coronary artery endothelial cells (HCAEC) adapted to prolonged moderate (250C) hypothermia in vitro have high levels of cellular glutathione, an extensively modified proteome and increased resistance to cold-induced oxidative stress and cell death at 00C, a temperature that is highly damaging. The objective of this R21 application is to determine how endothelial cold-adaptation leads to enhanced protection from 00C injury. The central hypothesis is that mild- moderate hypothermia induces a non-lethal endoplasmic reticulum (ER) stress and the ensuing response activates transcriptional pathways that are cytoprotective. The rationale for the proposed studies is that defining the mechanisms of cold-adaptation will provide the tools to make heart preservation safer and will yield better clinical outcomes. The central hypothesis will be tested by pursuing the following two specific aims: 1) Determine the effect of mild-moderate hypothermia on ER stress signaling;and 2) Determine the effect of mild- moderate hypothermia on the Nrf2/ARE pathway. The first aim will determine the temperature dependence, kinetics and extent of activation of the Unfolded Protein Response in HCAECs and then use this knowledge, UPR pathway inducers and RNAi inhibition of UPR mediators to modulate the expression of antioxidant proteins and chaperones, glutathione synthesis, and subsequent cell survival at 00C. In the second aim, temperature, induction/inhibition of ER stress, promotion/reduction of oxidative stress, and RNAi inhibition of Nrf2/Keap1 will be used to modulate the activation of the Nrf2/ARE pathway and thereby vary the expression of antioxidant proteins and glutathione synthesis and subsequent cell survival at 00C. The proposed research is innovative because it focuses on a mechanism of hypothermic adaptation in homeothermic mammalian cells rather than using more conventional approaches to organ preservation research. This is highly significant because it facilitates the study of novel protective mechanisms that would not be considered if one only studied the pathology of preserved organs. PUBLIC HEALTH RELEVANCE: The proposed studies are in an important area of research recently recommended by an NHLBI working group on future directions for research in cardiac surgery. The proposed research will establish a fundamentally new strategy for preserving the heart, which is the exploitation of molecular pathways involved in the inherent adaptability of cells to cold. Improved heart preservation methods will benefit not only heart transplant recipients, but also patients requiring surgical procedures, such as coronary artery bypass grafting or the correction of congenital heart defects.